CN103149312B - Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry - Google Patents

Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry Download PDF

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CN103149312B
CN103149312B CN201310045619.XA CN201310045619A CN103149312B CN 103149312 B CN103149312 B CN 103149312B CN 201310045619 A CN201310045619 A CN 201310045619A CN 103149312 B CN103149312 B CN 103149312B
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sialic acid
sialic
liquid chromatography
performance liquid
acid
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CN103149312A (en
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李春
刘宁
刘丽波
解鸿蕾
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Northeast Agricultural University
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Abstract

The ineveniton discloses an analyzing method of sialic acid in infant milk powders with an ultra-high performance liquid chromatography tandem quadrupole mass spectrometry method. On the basis of an original detecting of the sialic acid in milk powders with high performance liquid chromatography, the ultra-high performance liquid chromatography (UPLC) technology is combined, the sialic acid in three forms in a sample is extracted, and therefore the method of detecting the sialic acid in infant formula milk with the UPLC tandem mass spectrometry, wherein the method is higher in sensitivity and faster. According to the method, the average recovery of the sialic acid is within 84.3%-98.9%, the relative standard deviation (RSD) is 4.9%-8.2%, and the lowest detection limit of the sialic acid is 0.01 microgram/milliliter. The analyzing method of the sialic acid in infant milk powders with the UPLC tandem quadrupole mass spectrometry is simple and convenient to operate, a better separation mode is adopted, matrix effects are relatively small, the process of high-speed centrifugation after hydrolysis is adopted in pretreatment and simple, easy and convenient to operate, the loss of the sialic acid in a pretreatment process is effectively avoided, and the recovery rate is improved.

Description

One utilizes Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry to analyze sialic method in infant milk powder
Technical field
The present invention relates to a kind of sialic method in analysis infant milk powder, particularly one utilizes Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry to analyze sialic method in infant milk powder, belongs to field of food detection.
Background technology
Sialic acid, refers to a series of general designation containing the carboxylated monose acylated derivatives of nine carbon atoms, and its systematic naming method is amino-3, the 5-dideoxy-D-glycerine-D-gala ketononoses of 5-.It is in human milk and infant formula Ruzhong, combine and protein-bound form existence with free, compound sugar respectively, human milk oligosaccharides bound sialic acid accounts for 73%, infant formula milk protein combination sialic acid accounts for 70%, mainly N-acetyl-neuraminate (N-acetylneuraminic acid, Neu5Ac), so often N-acetyl-neuraminate is called sialic acid, cow's milk and infant formula Ruzhong sialic acid content all very low.Latest report proves that sialic acid is also a kind of natural brain nutrient, can promote memory and the intelligent development of infant, is paid close attention to widely with its high-load in breast milk, but content in milk and infant formula is very low.This also imply that in milk power for infant and young children, particularly effectively can promote their nervous system and the growth of brain for adding sialic acid in the milk power for infant and young children of premature, improves it simultaneously further and is growing early stage intelligent development level.
The method analyzed for sialic acid is a lot, and enzyme reaction, antibody, agglutinin and virus are for determining the sialic acid of Total silicic acid content or detection particular type; Orcin, resorcinol, regularly/thiobarbituricacidα-colourimetry, spectrophotometric method has also been widely used in sialic detection for many years.The common limitation of these methods is that they cannot distinguish sialic type and require sample purification.The classic methods such as colourimetry and chromatogram photometric method are then the sialic acids after using ion exchange column purification acid hydrolysis sample, and in this method sample, sialic acid loss is comparatively large, the accuracy that impact detects.Sialic detection method in food, bibliographical information has high performance liquid chromatography (HPLC), but its pre-treatment is loaded down with trivial details, and needs are measuring after asking and carrying out derivatization to sialic acid, add the complicacy of experiment, and sensitivity and accuracy not also very desirable.
Fewer due to what apply in sialic acid at home food, and the matrix that Ruzhong self exists, as protein, vitamin, calcium, potassium, the mineral matters such as magnesium can impact analysis result, are not therefore also very ripe about the correlation technique detected it.Therefore, study a kind of efficient method detecting sialic acid content in milk power for infant and young children fast, further optimization process is done for lower content sample, sialic content is improved in its prescription emulsifiable powder, make it closer to breast milk, thus better promoting the healthy development of infant's brain, the physical nutritional needs meeting infant growth become a kind of exigence.At present, home market has infant formula milk and adds sialic acid, makes it closer to human milk components.Along with the increase adding sialic acid food, food quality supervision department and manufacturing enterprise's quality control need also to be on the increase, certainly will require more accurately with detection method fast.Along with the development of chromatographic column filler technology, the Ultra Performance Liquid Chromatography (UPLC) based on 1.7 μm of granule technology has higher separating power than traditional HPLC, and mass spectrometry can carry out the Accurate Measurement of molecular weight.
Li Hui etc. utilize liquid chromatography-mass spectrography (Li Hui, Jin Yibao, Liu Hongxia, in managing state affairs, analytical test journal, 2008,11 (27): 193-194) sialic acid content in milk powder is measured, with tetrabutylammonium aqueous solution-methyl alcohol for mobile phase carries out gradient elution, triple quadrupole bar mass spectrum adopts negative ion mode, and detect and be limited to 0.06mg/L, relative standard deviation is less than 10%.The chromatogram applied and Mass Spectrometry Conditions as follows: chromatographic column: XBridgeC 18(3.5 μm, 2.1mm × 100mm) (Waters, Massachusetts, USA); Mobile phase: 4mmol/L tetrabutylammonium aqueous solution-methanol elution gradient; Flow velocity: 0.2mL/min; Sample size 10 μ L; Triple quadrupole bar mass spectrum adopts negative ion mode, and ion source temperature is 110 ° of C, and ion gun ionization voltage is 3kV, and atomization gas flow velocity is 800L/h -1, ion acquisition mode adopts multiple-reaction monitoring pattern (MRM), and gather ion 308.1>87.1, retention time is 6.1min.
The method utilizes the mass spectrographic method of liquid chromatogram-triple tandem quadrupole, adopt many reaction negative ion monitoring patterns, mobile phase selects tetrabutylammonium aqueous solution-methanol solution to carry out gradient elution, good separation is achieved to the sialic acid in milk powder, retention time is 6.1min, sialic minimum detectability is 0.06mg/L, and relative standard deviation is less than 10%.But be separated at mass spectrum, when application electric spray ion source is to parent ion full scan, we find, under negative ion multiple-reaction monitoring pattern, the Abundances of parent ion is not very high, for this reason bound sialic acid chemical structural formula we courageously suppose, positive ion multiple-reaction monitoring pattern can be applied completely full scan is carried out to the parent ion of sialic acid standard, find the reliable Mass Spectrometry Conditions that Abundances is higher under negative source and positive source condition, for the sensitivity and accuracy improving detection method lays the foundation; Secondly in conjunction with chromatographic column selective flow phase time, we think that mobile phase should make sialic acid be well separated on a column, best response is obtained in retention time, therefore numerous conforming to the principle of simplicity to be abandoned when studying eluent gradient wash-out, select in excellent and excellently determine mobile phase and proportioning thereof, and then improve sensitivity and the detection limit of this method, do basis for setting up industry standard.
Feng Jun etc. adopt high performance liquid chromatography to carry out measuring (Feng Jun, Li Hongji, Han Liqiang to the sialic content in Ruzhong, Li Weihua, Li Ping, Yang Guoyu, modern food science and technology, 2007,12:23), utilize acid-hydrolysis method that the sialic acid in Ruzhong is discharged, with o-dihydroxy ammon hydrochloride (10mg/mL) for derivatization reagent, in 80 DEG C of water-baths, derivative 40min, adopts Symmetry C 18post (250mm × 4.6mm, 5 μm); Mobile phase is tetrahydrofuran aqueous solution (containing 0.2% the phosphoric acid)-acetonitrile (92:8) of 1.0%; Flow velocity is 1.0mL/min, and column temperature is 35 DEG C; UV detect wavelength 230nm.Result shows, sialic acid is good in 10 ~ 320 μ g/mL scope internal linear, and the lowest detection of N-acetyl-neuraminate is limited to 0.25 μ g/mL, and average recovery rate is 95.7%.
The method utilizes conventional high performance liquid chromatography, because most saccharide compound does not have chromophoric group, sialic acid is no exception, so the analysis of HPLC needs to carry out derivative rear separation determination to sialic acid, adds the complicacy of experiment and possible disturbing factor.Derivatization reagent is economically considered relatively costly, and derivatization process relatively bothers, loaded down with trivial details; Sensitivity and the accuracy of high performance liquid chromatography detection are simultaneously limited, therefore research method should try every possible means to prevent the loaded down with trivial details of pre-treatment, reduce the complicacy of experiment and possible disturbing factor, determine the simplest Pretreatment of optimization further, adopt rational separation and detection technology, and then improve sialic detection technique and standard in Milk Powder Formula For Infants.Although the method can Accurate Determining Ruzhong sialic acid content, sensitivity and accuracy need further raising.
Summary of the invention
For Problems existing in currently available technology, the present invention to detect in milk powder on sialic basis in original high performance liquid chromatography, based on 1.7 μm of short grained Ultra Performance Liquid Chromatography (UPLC) technology, three kinds of form sialic acids in sample are extracted, thus establish sensitivity higher, faster Ultra Performance Liquid Chromatography tandem mass spectrum detect baby formula Ruzhong sialic method, actually detect sample successful.
In order to reach above object, the technological means that the present invention adopts is:
One of the present invention utilizes Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry to analyze sialic method in Milk Powder Formula For Infants, it is characterized in that comprising the following steps:
(1) drawing standard curve;
(2) pre-treatment of sample: three kinds of form sialic acids in sample are extracted, three kinds of described form sialic acids are respectively free sialic acid, compound sugar bound sialic acid and protein combination sialic acid;
Extract the Solid-Phase Extraction column purification that the three kinds of form sialic acid solution obtained adopt HLB polymer filler respectively, adopt ultrapure water as leacheate, ammonia water-methanol mixed solution is as eluent;
(3), after the extract solution mixing after purification step (2) obtained, adjust ph to 2.5 ~ 3, use acetonitrile constant volume, and 0.22 μm of organic membrane filtration, measures with UPLC-ESI-MS/MS;
(4) selection of mass spectrophotometry condition and parameter:
Ion gun: electric spray ion source; Scan mode: positive ion scans; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 DEG C; Desolventizing temperature: 450 DEG C; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Impinging air flows amount: 0.24mL/min; Photomultiplier cell voltage: 650V;
(5) chromatographic separation condition:
Chromatographic column and gradient elution: specification is 50mm × 2.1mm, the Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post of 1.7 μm; Column temperature: 30 DEG C; Sample chamber temperature: 30 DEG C; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1% formic acid (v/v) aqueous solution.
In the present invention, preferably, three kinds of form sialic acids described in step (2) extract in accordance with the following methods:
The extraction of free and compound sugar bound sialic acid: precision takes a certain amount of Milk Powder Formula For Infants, adding solution of trichloroacetic acid to dissolving completely, reaching the object of protein precipitation, getting supernatant, 4 DEG C, under 15000r/min condition, centrifugal 10min, for subsequent use;
The sialic extraction of protein combination: add hydrochloric acid solution in the precipitation after the extraction of compound sugar bound sialic acid " free and " process, put into 80 DEG C of water-bath 1h, cooling after taking out, 4 DEG C, under 15000r/min condition, centrifugal 10min, for subsequent use.
Wherein, preferably, the concentration of described solution of trichloroacetic acid is 20%(v/v), described solution of trichloroacetic acid (ml) is 1:6-10 with the volume mass ratio of Milk Powder Formula For Infants (g); The concentration of described hydrochloric acid solution is 0.1%(v/v), described hydrochloric acid solution (ml) is 1:2-5 with the volume mass ratio of Milk Powder Formula For Infants (g).
In the present invention, preferably, the solid-phase extraction column of the HLB polymer filler described in step (2) is in Oasis HLB solid phase extraction column, and in ammonia water-methanol mixture, the volume ratio of ammoniacal liquor and methyl alcohol is 25:75.
In the present invention, preferably, the 2-5 of former extract mixed liquor volume is settled to doubly with acetonitrile in step (3).
In the present invention, preferably, the desolventizing gas described in step (4) is nitrogen, and described collision gas is argon gas.
In the present invention, preferably, the elution program of the gradient elution described in step (5) is: in initial wash liquid, the percent by volume of mobile phase A is 90%, and the percent by volume of Mobile phase B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A are down to 12%, B by 90% and are upgraded to 88% by 10%, and are maintained until 1.8min; 1.8 ~ 2.2min, A are upgraded to 90%, B by 12% and are down to 10% by 88%.
Experiment proves, it is 84.3% ~ 98.9% that the standard using the inventive method to measure adds average recovery rate, and sialic minimum detectability is 0.01 μ g/mL, and the method that known the present invention sets up has the higher recovery and sensitivity.
Compared to prior art, the invention has the advantages that:
1, establish more quick, sensitivity and the higher Ultra Performance Liquid Chromatography tandem mass spectrometry of accuracy detect the sialic method in Milk Powder Formula For Infants;
2, adopt multiple-reaction monitoring pattern, negative ion scan mode, determines Mass Spectrometry Conditions, and then the accuracy of raising method;
3, when chromatographic resolution, by choosing the analysis of chromatographic column, determine to adopt BEH HILIC (50mm × 2.1mm, 1.7 μm) chromatographic column, choose 0.1% aqueous formic acid and acetonitrile as mobile phase, adopt gradient elution mode, guaranteeing the successful reservation in sialic chromatographic column, laying the foundation for analyzing its content further.
This product basic mechanical design feature index:
1, sialic acid is good with the linear relationship of sialic acid peak area within the scope of 0.05 ~ 5.0 μ g/mL;
2, sialic average recovery rate is between 84.3% ~ 98.9%, and relative standard deviation is 4.9% ~ 8.2%;
3, sialic lowest detection is limited to 0.01 μ g/mL.
Accompanying drawing explanation
Fig. 1 is the MRM chromatogram of typical 1.0 μ g/mL sialic acid standard;
Fig. 2 is the MRM chromatogram of blank powdered milk sample;
Fig. 3 is the MRM chromatogram of mark-on powdered milk sample.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The mensuration of sialic acid content in embodiment 1 Milk Powder Formula For Infants
Method:
1, the drafting of typical curve
Accurately pipette appropriate sialic acid standard storing solution, dilution obtains the standard solution that concentration is 0.05,0.1,0.2,0.5,1.0 and 5.0 μ g/mL successively.Carry out linear regression, drawing standard curve according to the peak area (Y) that sialic quota ion measures with sialic mass concentration (X, μ g/mL), sialic equation of linear regression is:
Y=8514.5X－811.09，R 2=0.9989
Wherein Y is the chromatographic peak peak area measured, and X is sialic mass concentration (μ g/mL), learns that the linear relationship of this typical curve is good.
2, the pre-treatment of sample
Get each 1.0g of milk power for infant and young children sample A1, B2, C3 of 3 parts of different brands, carry out three kinds of sialic extractions of form respectively in accordance with the following methods, each sample 3 parallel analysiss, upper machine carries out determination experiment, carries out quantitatively with matrix-matched curve.
The extraction of 2.1 free and compound sugar bound sialic acids
Precision takes 1.0g milk power for infant and young children, add 8mL, 20% solution of trichloroacetic acid to dissolving completely, reach the object of protein precipitation, getting supernatant loads in the centrifuge tube of 4 2mL, 4 DEG C, under 15000r/min condition, centrifugal 10min, merges supernatant for subsequent use.
The sialic extraction of 2.2 protein combination
Add 2mL, 0.1%(v/v in precipitation after the extraction of compound sugar bound sialic acid " free and " process) hydrochloric acid solution, put into 80 DEG C of water-bath 1h, cooling after taking out, 4 DEG C, under 15000r/min condition, centrifugal 10min.
The purification of 2.3 sample supernatant
The centrifugal sample solution obtained is loaded into Oasis HLB solid phase extraction column (Beijing Zhen Xiang company, 3mL/60mg), experiment adopts ultrapure water as leacheate, ammonia water-methanol (volume ratio 25:75) solution is as eluent, and wherein 4mL leacheate and 3mL eluent can meet purification requirement.
2.4, finally after two parts extract solution mixing after purification, about adjust ph to 2.5 ~ 3, are settled in 20mL volumetric flask with acetonitrile, with 0.22 μm of organic membrane filtration, measure with UPLC-ESI-MS/MS.
The selection of 3, mass spectrophotometry condition and parameter
Ion gun: electric spray ion source (ESI); Scan mode: positive ion (ESI+) scans; Detection mode: multiple-reaction monitoring system (MRM); Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 DEG C; Desolventizing temperature: 450 DEG C; Desolventizing gas (nitrogen) flow: 650L/hr; Taper hole blowback air flow: 50L/hr; Collision gas (argon gas) flow: 0.24mL/min; Photomultiplier cell voltage: 650V;
4, Ultra Performance Liquid Chromatography condition
Chromatographic column: Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post (Waters, US) (50mm × 2.1mm, 1.7 μm); Column temperature: 30 DEG C; Sample chamber temperature: 30 DEG C; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1% aqueous formic acid; Gradient elution program: original ratio A is 90%, B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A are down to 12%, B by 90% and are upgraded to 88% by 10%, and are maintained until 1.8min; 1.8 ~ 2.2min, A are upgraded to 90%, B by 12% and are down to 10% by 88%.
Result:
The confirmatory analysis of actual sample:
Get each 1.0g of milk power for infant and young children sample A1, B2, C3 of 3 parts of different brands, after the method process of setting up, each sample 3 parallel analysiss, upper machine carries out determination experiment, carry out quantitatively with matrix-matched curve, the sialic content obtaining 3 parts of different samples is respectively 107.7 μ g/g, 72.9 μ g/g and 55.8 μ g/g.With existing method (sialic assay method in the milk powder such as yellow Hua Jun, Xi Xinglin. analytical test journal, 2006,25 (4): 129 ~ 131; Sialic acid content in the high effective liquid chromatography for measuring breast milks such as Chen Haijiao, Wang Ping. Food Science, 2011,32 (16): 1002 ~ 1008) measurement result makes comparisons, and learns that the sample size of checking the method is in rational scope.
The selection of test example 1 mass spectrophotometry condition and the optimization of parameter
Get sialic standard solution (mass concentration is 0.5 μ g/mL), under ESI bears source and positive source condition, full scan is carried out to parent ion.Sialic parent ion bar graph under obtaining different ions source.Experimental result shows: the abundance of sialic acid parent ion bar graph under the condition of ESI positive source more than the height in negative source, therefore selected the positive source of ESI to do ionization source.
Under positive source module, parent ion full scan is carried out to sialic standard solution (mass concentration is 0.5 μ g/mL), determine various sialic quasi-molecular ion and optimize taper hole voltage (cone voltage respectively, CV), determine that final taper hole voltage is 45V; Then with its quasi-molecular ion for parent ion, parent ion is 310.1, carries out full scan to its daughter ion, and select abundance is higher in fragmention two ions as qualitative, quantitative characteristic ion, daughter ion is 274.1 and 292.1, and wherein 274.1 is quota ion.Sialic acid quantitative and qualitative analysis ion current chromatogram is shown in Fig. 1.
Last with the various mass spectrum tuner parameters of positive ion multiple-reaction monitoring model-based optimization, farthest improve detection sensitivity, the results are shown in Table 1.
The sialic chromatography-mass spectroscopy parameter of table 1
*: feature daughter ion
The mass spectrophotometry condition finally determined and parameter:
Ion gun: electric spray ion source; Scan mode: positive ion scans; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 DEG C; Desolventizing temperature: 450 DEG C; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Impinging air flows amount: 0.24mL/min; Photomultiplier cell voltage: 650V.
The optimization of test example 2 chromatographic separation condition
The selection of chromatographic column:
The physicochemical property of bound sialic acid and sialic acid, at the partition characteristic of chromatographic column, select the Waters ACQUITY Ultra BEH HILIC(2.1 × 50mm and 2.1 × 100mm based on 1.7 μm of granule technology respectively), BEH C 18with phenyl post four kinds of chromatographic columns, sialic acid is separated, learns that sialic retention time has larger difference.Adopt BEH C 18be separated with phenyl post, after optimizing, sialic retention time is respectively 0.51min and 0.60min, and retention time is too short, and when standard items and sample detection, signal strength differences is comparatively large, and sample sensitivity is all low, is unsuitable for sample detection.Adopt BEH HILIC(2.1 × 100mm), after optimizing, sialic retention time is 2.15min, and retention time is comparatively reasonable, but the hangover of peak type, chromatographic behavior is undesirable, and sensitivity is lower.Finally select BEH HILIC(2.1 × 50mm, 1.7 μm) carry out determination and analysis, find that this post has good separating effect and higher sensitivity to sialic acid, retention time 1.22min is optimum value, illustrate that this post has more good separating power than traditional chromatographic column, response and sensitivity all can reach the requirement of experiment, and blank sample and mark-on sample drawing are shown in shown in Fig. 2 and Fig. 3.
The selection of mobile phase:
Sialic acid is a kind of carbohydrates, usually exists with the form of glucosides with free state or at the end of glycoprotein or glycolipid, belongs to carbohydrate.Add 90% acetonitrile test with 10%5mM formic acid ammonia solution, water, 0.1% formic acid solution respectively in experiment, with standard items sample introduction under different mobile phase condition of 1.0 μ g/mL, when result shows that 0.1% formic acid solution makes mobile phase, signal is the strongest, chromatographic behavior is better, and retention time is desirable.And acetonitrile has excellent solvent nature because of it, polarity is comparatively large, can dissolve most sample, infinitely dissolve each other with water and alcohol, and can improve the peak type of chromatographic peak.Therefore, mobile phase selects 0.1% aqueous formic acid and acetonitrile.Contrast in isocratic elution and gradient elution, when finding isocratic elution, chromatogram peak-to-peak type trails, undesirable.Finally determine to choose 0.1%(v/v) aqueous formic acid and acetonitrile be as mobile phase, adopt the mode of gradient elution thus realize sialic good separation, significantly improve sialic acid distribution in the chromatography column, and there is higher Ionization Efficiency, strengthen the sensitivity detected.Sialic acid gradient elution separable programming is as shown in table 2.
Table 2UPLC/MS/MS sialic acid gradient elution separable programming
Original ratio A is 90%, B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A are down to 12%, B by 90% and are upgraded to 88% by 10%, and are maintained until 1.8min; 1.8 ~ 2.2min, A are upgraded to 90%, B by 12% and are down to 10% by 88%.
The optimization of test example 3 Pretreatment
The optimization of extraction conditions:
From different material, the sialic method of separation and Extraction is had nothing in common with each other, and after sialic acid release, most ion-exchange chromatography that adopts is separated with gel permeation chromatography and utilizes the steps such as HPLC purifying.Because the good chromatographic column of separating effect is selected in this experiment, pre-treatment have employed the rear ultracentrifugal process of hydrolysis, and method is easy, effective.The focus paid close attention in hydrolysis procedures is that carbohydrates is subject to minimum destruction and sialic maximum release, research shows, with 0.1%(v/v under 80 DEG C of conditions) hydrochloric acid hydrolysis 1h, all sialic acid chains can be made to rupture and make these residue deacylated tRNA decarboxylation, ensureing sialic complete hydrolysis.
The optimization of purification condition:
Because the comparison of ingredients in milk powder is complicated, the supernatant after centrifugal need carry out Solid-Phase Extraction to remove the impurity in sample liquid.The solid phase extraction method that the sample of current milk powder class is conventional mainly adopts anti-phase and ion-exchange SPE post, also has HLB solid-phase extraction column.Research shows, adopts the Solid-Phase Extraction column purification of HLB polymer filler, can make the clean-up effect that sample reaches good, and method is simple, favorable reproducibility.Be loaded into by sample solution in Oasis HLB solid phase extraction column, experiment adopts ultrapure water as leacheate, and ammonia water-methanol (volume ratio 25:75) solution is as eluent, and wherein 4mL leacheate and 3mL eluent can meet purification requirement.
Test example 4 Method validation
Linear relationship is investigated:
Choose not containing the blank powdered milk sample of component to be measured, premenstrual disposal methods prepares bare substrate liquid, accurately pipette appropriate sialic acid standard storing solution, add in bare substrate liquid, join the extraction standard solution that concentration is followed successively by 0.05,0.1,0.2,0.5,1.0 and 5.0 μ g/mL.Upper machine measures, record peak area.The peak area (Y) measured according to sialic quota ion and sialic mass concentration (X, μ g/mL) carry out linear regression, drawing standard curve, through Masslynx v4.1 data processing software analyzing and processing, sialic equation of linear regression is:
Y=8114.5X－811.09，R 2=0.9989
Wherein Y is the chromatographic peak peak area measured, and X is sialic mass concentration (μ g/mL).
The recovery, precision and detection limit:
Take the infant milk powder sample of the unknown sialic acid concentration of 1.0g, measure according to after the method process of embodiment 1, another title 1.0g sample is on pre-treatment basis, get the supernatant of about 7mL, and add the standard solution that concentration is 5.0 μ g/mL, with acetonitrile constant volume in the volumetric flask of 10mL, upper machine measures, the selection of mass spectrophotometry condition and parameter, Ultra Performance Liquid Chromatography condition set described in embodiment 1, obtain sialic concentration in unknown sample.On the basis of former content sample, add the sialic acid standard specimen of 0.1,0.5,2.5 and 5.0 μ g/mL successively respectively, deduct blank after detection, the recovery of assay method, data are as shown in table 3.
The sialic TIANZHU XINGNAO Capsul of table 3, precision and detection limit (LOD) (n=3)
It is 84.3% ~ 98.9% that this method adds average recovery rate at bioassay standard, has the good recovery; The range of linearity lower limit of this method is 0.05 μ g/mL, and its signal to noise ratio (S/N ratio) (S/N) is greater than 10, therefore determines quantitatively to be limited to 0.05 μ g/mL; With the detection limit of 3 times of S/N computing method, learn that sialic minimum detectability is 0.01 μ g/mL, the method for known foundation has higher sensitivity.

Claims (6)

1. utilize Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry to analyze a sialic method in Milk Powder Formula For Infants, it is characterized in that comprising the following steps:
(1) drawing standard curve;
(2) pre-treatment of sample: three kinds of form sialic acids in sample are extracted, three kinds of described form sialic acids are respectively free sialic acid, compound sugar bound sialic acid and protein combination sialic acid;
Extract the Solid-Phase Extraction column purification that the three kinds of form sialic acid solution obtained adopt HLB polymer filler respectively, adopt ultrapure water as leacheate, ammonia water-methanol mixed solution is as eluent;
(3), after the extract solution mixing after purification step (2) obtained, adjust ph to 2.5 ~ 3, use acetonitrile constant volume, and 0.22 μm of organic membrane filtration, measures with UPLC-ESI-MS/MS;
(4) selection of mass spectrophotometry condition and parameter:
Ion gun: electric spray ion source; Scan mode: positive ion scans; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 DEG C; Desolventizing temperature: 450 DEG C; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Impinging air flows amount: 0.24mL/min; Photomultiplier cell voltage: 650V;
(5) chromatographic separation condition:
Chromatographic column and gradient elution: specification is 50mm × 2.1mm, the Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post of 1.7 μm; Column temperature: 30 DEG C; Sample chamber temperature: 30 DEG C; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1%v/v aqueous formic acid; The elution program of gradient elution is: in initial wash liquid, the percent by volume of mobile phase A is 90%, and the percent by volume of Mobile phase B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A are down to 12%, B by 90% and are upgraded to 88% by 10%, and are maintained until 1.8min; 1.8 ~ 2.2min, A are upgraded to 90%, B by 12% and are down to 10% by 88%.
2. in accordance with the method for claim 1, it is characterized in that three kinds of form sialic acids described in step (2) extract in accordance with the following methods:
The extraction of free and compound sugar bound sialic acid: precision takes a certain amount of Milk Powder Formula For Infants, adding solution of trichloroacetic acid to dissolving completely, reaching the object of protein precipitation, getting supernatant, 4 DEG C, under 15000r/min condition, centrifugal 10min, for subsequent use;
The sialic extraction of protein combination: add hydrochloric acid solution in the precipitation after the extraction of compound sugar bound sialic acid " free and " process, put into 80 DEG C of water-bath 1h, cooling after taking out, 4 DEG C, under 15000r/min condition, centrifugal 10min, for subsequent use.
3. in accordance with the method for claim 2, it is characterized in that the concentration of described solution of trichloroacetic acid is 20%v/v, described solution of trichloroacetic acid is 1:6-10 with the volume mass ratio of Milk Powder Formula For Infants; The concentration of described hydrochloric acid solution is 0.1%v/v, and described hydrochloric acid solution is 1:2-5 with the volume mass ratio of Milk Powder Formula For Infants.
4. in accordance with the method for claim 1, it is characterized in that the solid-phase extraction column of the HLB polymer filler described in step (2) is in Oasis HLB solid phase extraction column, in ammonia water-methanol mixture, the volume ratio of ammoniacal liquor and methyl alcohol is 25:75.
5. in accordance with the method for claim 1, it is characterized in that being settled to the 2-5 of former extract mixed liquor volume doubly with acetonitrile in step (3).
6. in accordance with the method for claim 1, it is characterized in that the desolventizing gas described in step (4) is nitrogen, described collision gas is argon gas.
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